• Proceedings of the Korean Information Science Society Conference
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• 2006.06a
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• pp.25-27
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• 2006

본 논문에서는 시계열 마이크로어레이데이터 마이닝을 위한 전처리 작업으로 시계열 마이크로어레이 데이터에 특징 추출 방법 및 상관관계 분석을 이용하여 분화 과정에 대해 분별력 있는 유전자들을 선정하기 위한 방법을 제안하고, 줄기세포가 신경세포로 분화하는 과정에서 특이적으로 발현되는 유전자들을 찾기 위한 시계열 마이크로어레이 데이터 분석 과정을 하나의 예로 제시한다. 분석 결과, 제안한 방법이 분화 특이적으로 발현되는 분별력 있는 유전자들, 분화 과정에서 공통적으로 발현되는 유전자들, 그리고 경계선에 존재하는 유전자들을 통해서 줄기세포 신경분화의 특징들을 규명하는데 매우 유용함을 보였다.

• Development and Reproduction
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• v.12 no.1
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• pp.31-39
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• 2008

Neural tissue has limited intrinsic capacity of repair after injury, and the identification of alternate sources of neural stem cells has broad clinical potential. We isolated mesechymal-like stem cells from human adipose tissues (AT-MSCs), and studied on transdifferentiation-promoting conditions in neural cells. Dopaminergic and cholinergic neuron induction of AT-MSCs was also studied. Neural differentiation was induced by adding bFGF, EGF, dimethyl sulphoxide (DMSO) and butylated hydroxyanisole(BHA) in N2 Medium and N2 supplement. The immunoreactive cells for $\beta$-tubulin III, a neuron-specific marker, GFAP, an astrocyte marker, or Gal-C, an oligodendrocyte marker, were found. AT-MSCs treated with bFGF, SHH and FGF8 were differentiatied into dopaminergic neurons that were immunopositive for TH antibody. Differentiation of MSCs to cholinergic neurons was induced by combined treatment with basic fibroblast growth factor (bFGF), retinoic acid (RA) and sonic hedgehog (Shh). AT-MSCs treated with DMSO and BHA rapidly assumed the morphology of multipolar neurons. Both immunocytochemistry and RT-PCR analysis indicated that the expression of a number of neural markers including neuro D1, $\beta$-tubulin III, GFAP and nestinwas markedly elevated during this acute differentiation. While the stem cell markers such as SCF, C-kit, and Stat-3 were not expressed after preinduction medium culture, we confirmed the differentiation of dopaminergic and cholinergic neurons by TH/$\beta$-tubulin III or ChAT/ $\beta$-tubulin III positive cells. Conclusively, AT-MSCs can be differentiated into dopaminergic and cholinergic neuronsand these findings suggest that AT-MSCs are alternative cell source of treatment for neurodegenerative diseases.

• Journal of Life Science
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• v.32 no.2
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• pp.108-118
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• 2022

The subventricular zone (SVZ) in the brain contains neural stem cells (NSCs) that generate new neurons throughout one's lifetime. Many extracellular and intracellular factors that affect cell proliferation and neuronal differentiation of NSCs are already well-known. Recently, L-type calcium channels have been reported to regulate neural development and are present in NSCs, differentiating neuroblasts, and mature neurons in the SVZ. Nifedipine, a blocker of L-type calcium channels, has been long used as a therapeutic drug for hypertension. However, studies on the use of nifedipine to inhibit L-type calcium channels of NSCs are lacking. Herein, we treated NSCs cultured from mouse postnatal SVZ with nifedipine during neuronal differentiation. Nifedipine increased the number of Tuj1-positive neurons but did not significantly change the number of Olig2-positive oligodendrocytes. Nifedipine increased cell division during early differentiation, which was detected using the 5-ethynyl-2'-deoxyuridine incorporation assay and immunocytochemistry assessment by staining the cells with phosphorylated histone H3, a mitosis marker. Nifedipine increased the transcription of Dlx2, a neurogenic transcription factor, and the level of Mash1, a marker for early neurogenesis. In addition to nifedipine, verapamil, which is also an L-type calcium channel blocker, showed a slight increase in neurogenesis, but its statistical significance was very low. In contrast, pimozide, a T-type calcium channel blocker, did not affect neurogenesis, although T-type calcium channel genes Cav3.1, Cav3.2, and Cav3.3 were expressed. In summary, nifedipine might promote the neuronal fate of NSCs during early differentiation and calcium signaling through L-type calcium channels might be involved in neuronal differentiation, especially during the early stages of differentiation.

• Journal of Life Science
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• v.28 no.12
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• pp.1397-1405
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• 2018

Mitochondria have multiple functions in cells: providing chemical energy, storing cellular $Ca^{2+}$, generating reactive oxygen species, and regulating apoptosis. Through these functions, mitochondria are also involved in the maintenance, proliferation, and differentiation of stem/progenitor cells. In the brain, the subventricular zone (SVZ) is one of the neurogenic regions that contains neural stem cells (NSCs) throughout a lifetime. However, reports on the role of mitochondria in SVZ NSCs are scarce. Here, we show that rotenone, a complex I inhibitor of mitochondria, inhibits the proliferation and differentiation of SVZ NSCs in different ways. In proliferating NSCs, rotenone decreases mitosis as measured through phosphorylated histone H3 detection; moreover, apoptosis is not induced by rotenone at 50 nM. In differentiating NSCs, rotenone blocks neurogenesis and oligodendrogenesis while glial fibrillary acidic protein-positive astrocytes are not affected. Interestingly, in this study there were more cells in the differentiating NSCs treated with rotenone for 4-6 days than in the vehicle control group which was a different effect from the reduced number of cells in the proliferating NSCs. We examined both apoptosis and mitosis and found that rotenone decreased apoptosis as detected by staining cleaved caspase-3 but did not affect mitosis. Our results suggest that functional mitochondria are necessary in both the proliferation and differentiation of SVZ NSCs. Furthermore, mitochondria might be involved in the mitosis and apoptosis that occur during those processes.

• Journal of Life Science
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• v.12 no.3
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• pp.306-316
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• 2002

The ultrastructural change of sciatic nerve and immunohistochemical changes of NGF, PCNA were studied at the transplanted segment of intravascular cultured neural stem cell in the rat sciatic nerve by 5 months after the sciatic nerve transection. The transplanted intravascular neural stem cells were differentiated into Schwann reals at the 20th day and these cells began to regenerate by the proliferation and hypertrophy. There were many remyelinating Schwann cells in the transplanted nerve in term of stimulation. According to NGF finding, we suggest preexisting Schwann cells may induce the differentiation of neural stem cells into regenerating Schwann cells. Electron microscopic changes were the remyelinating appearance, the increase of intraaxonal microtubules and enlarged mitochondria and contacting tell processes each other.

• Proceedings of the Korean Information Science Society Conference
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• 2000.10b
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• pp.12-14
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• 2000

기존 진화 신경망 연구는 마지막 세대에서 최적의 신경망을 찾는 연구가 대부분이었다. 하지만 이 방법은 마지막 세대의 다른 신경망들이 진화와 학습을 통해 얻은 정보를 모두 무시한다. 최근에는 가능한 많은 정보를 이용해서 보다 뛰어난 성능의 시스템을 구축하기 위해, 마지막 세대의 모든 신경망들의 정보를 결합하는 다중 신경망에 관한 연구가 진행되고 있다. 효과적인 다중 신경망을 구축하기 위해서는 다양한 신경망들이 다중 신경망을 구성해서 서로 보완해 주도록 하여야 하는데, 아직까지 효과적인 다중 신경망 구축 방법은 나오지 않고 있다. 본 연구는 유전자 연산에서 다양한 해를 찾기 위해 사용하는 종분화를 이용해서 다양한 신경망들이 생성되도록 하는 다중 신경망 구축방법을 제안하고 실험을 통해 이 방법의 효용성을 보인다.

• Development and Reproduction
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• v.12 no.1
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• pp.87-95
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• 2008

Neural stem/precursor derived from pluripotent human embryonic stem cells (hESCs) has considerable therapeutic potential due to their ability to generate various neural cells which can be used in cell-replacement therapies for neurodegenerative diseases. However, production of neural cells from hESCs remains technically very difficult. Understanding neural-tube like rosette characteristic neural precursor cells from hESCs may provide useful information to increase the efficiency of hESC neural differentiation. Generally, neural rosettes were derived from differentiating hEBs in attached culture system, however this is time-consuming and complicated. Here, we examined if neural rosettes could be formed in suspension culture system by bypassing attachment requirement. First, we tested whether the size of hESC clumps affected the formation of human embryonic bodies (hEBs) and neural differentiation. We confirmed that hEBs derived from $500{\times}500\;{\mu}m$ square sized hESC clumps were effectively differentiated into neural lineage than those of the other sizes. To induce the rosette formation, regular size hEBs were derived by incubation of hESC clumps($500{\times}500\;{\mu}m$) in EB medium for 1 wk in a suspended condition on low attachment culture dish and further incubated for additional $1{\sim}2$ wks in neuroectodermal sphere(NES)-culture medium. We observed the neural tube-like rosette structure from hEBs after $7{\sim}10$ days of differentiation. Their identity as a neural precursor cells was assessed by measuring their expressions of neural precursor markers(Vimentin, Nestin, MSI1, MSI2, Prominin-1, Pax6, Sox1, N-cadherin, Otx2, and Tuj1) by RT-PCR and immunofluorescence staining. We also confirmed that neural rosettes could be terminally differentiated into mature neural cell types by additional incubation for $2{\sim}6$ wks with NES medium without growth factors. Neuronal(Tuj1, MAP2, GABA) and glial($S100{\beta}$ and GFAP) markers were highly expressed after $2{\sim}3$ and 4 wks of incubation, respectively. Expression of oligodendrocyte markers O1 and CNPase was significantly increased after $5{\sim}6$ wks of incubation. Our results demonstrate that rosette forming neural precursor cells could be successfully derived from suspension culture system and that will not only help us understand the neural differentiation process of hESCs but also simplify the derivation process of neural precursors from hESCs.

• Development and Reproduction
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• v.13 no.4
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• pp.227-237
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• 2009

Recently, adipose mesenchymal stem cells (AdMSC) that are similar to bone marrow MSC and blood derived MSC are thought to be another source for stem cell therapy. However, the diseases that can be applied for stem cells therapy are age-dependent degenerative diseases. Accordingly, the present study investigated the growth and differentiation potential to neural cells of human AdMSC (hAdMSC) obtained from aged thirty, forty and fifty. The growth of cells and cell viability were measured by passage and neural differentiation of hAdMSC was induced in neural differentiation condition for 10 days. Our results demonstrated that cell number, viability and morphology were not different from hAdMSC by age and passage. Immunofluorescence analysis of neural cell marker (TuJ1, NSE, Sox2, GFAP or MAP2) demonstrated no significant differences in neural cell differentiation by age and passage. As the number of passage was increased, the mRNA level of MAP2 and Sox2 was decreased in hAdMSC from age of 50 compared to hAdMSC from age of 30. In conclusion, the present study demonstrated that ability of neural cell differentiation of hAdMSC was maintained with ages, suggesting that autologous stem cells from aged people can be applied for stem cell therapy with age-dependent neural disease with the same stem cell quality and ability as stem cell derived from young age.

• Journal of Korean Ophthalmic Optics Society
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• v.18 no.4
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• pp.541-548
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• 2013

Purpose: Marrow stromal cells (MSCs) have been known for their potential to trans-differentiate into neural and glial cells in vitro and in vivo. To investigate the influence of the developing host environment on the survival and morphological and molecular differentiation, murine MSCs transplanted into the eye of Brazilian opossum (Monodelphis domestica). Methods: Enhanced green fluorescent protein (GFP) - expressing MSCs were transplanted into developing Brazilian opossums. Animals were allowed to survive for up to 4 weeks after transplantation, at which time the eyes were prepared for immunohistochemical analysis. Results: Some transplanted MSCs survived and showed morphological differentiation into neural cells with some processes within the host vitreous chamber. Some transplanted cells expressed class III ${\beta}$-tubulin (TuJ1, a marker for neuronal cells) or glial fibrillary acid protein (GFAP, a marker for glial cells) or Nestin (a marker for neural stem cells). In addition, some transplanted cells were located in ganglion cell layer but did not show morphological and molecular differentiation. Conclusions: Our result show that the most effective stage of development for transplantation into the retina was postnatal day 16, which retinas developmentally corresponded to postnatal day 4-5 days mouse retina based on cell differentiation and lamination patterns. The present findings suggest that the age of the host appears to play a key role in determining cell fate in vivo.

• Proceedings of the Korean Vacuum Society Conference
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• 2013.02a
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• pp.599-599
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• 2013

최근의 원자간력현미경(AFM)은 생체물질을 대상으로 여러 구조적 형상뿐만 아니라 물리적 특성 측정이 가능하여 바이오분야에 다양이 활용되고 있다. 줄기세포의 신경세포로 분화 인지에 대한 연구와 관련하여 본 연구에서는 AFM의 한 기능인 Force-Distance curve 측정법을 활용하여 신경암세포주라 불리는 SH-SY5Y를 대상으로 분화 전과 후의 세포막의 stiffness 변화를 측정하였다. 세포막의 stiffness값은 시료표면과 맞닿은 AFM 탐침에 계속적으로 수직방향의 힘이 가해질 시 AFM 캔티레버의 구부러짐 정도로 측정된다. SH-SY5Y는 RA (retinoic acid) 처리에 의해 분화유도 되었으며, 생물학적 방법인 western blotting법을 통해 분화여부를 확인하였다. 측정영역은 AFM topography 이미지 상에서 roughness가 가장 낮은 분화 전과 후 SH-SY5Y의 핵 주변영역으로 선정하였다. 선정된 영역 내에 여러 부분의 분화 전후 세포막의 stiffness 값을 측정하여 통계화한 결과, 분화 전과 후 세포막의 stiffness 차이를 확인할 수 있었다. 분화 전 SH-SY5Y 세포막의 stiffness는 0.79445 N/m인 반면, 분화 후 SH-SY5Y 세포막의stiffness는 0.60324 N/m로 확인되었다. 이는 분화 전에 비하여 분화 후 SH-SY5Y 세포막의 stiffness가 약 24.07% 감소된 것으로 판단할 수 있다. 본 연구는 생물학적 복잡한 방법이 아닌 간단한 방법으로 세포의 stiffness의 변화 측정을 통한 세포의 분화를 판별할 수 있는 방법을 개발한 것으로 여러 줄기세포의 특정세포로 분화여부 판단에 활용할 수 있을 것으로 사료된다.